Functionalized 9-bromo-camptothecin derivatives

ABSTRACT

The present invention relates to new functionalized 9-bromo-camptothecin derivatives (I) which have cytotoxic activity and are useful in treating diseases such as cancer, cellular proliferation disorders and viral infections. The present invention also provides methods for preparing these compounds, pharmaceutical compositions comprising them and methods of treating diseases utilizing such compounds or the pharmaceutical composition containing them. The invention also relates to the use of these functionalized 9-bromo-camptothecin derivatives in the preparation of conjugates.

The present invention relates to new functionalized 9-bromo-camptothecinderivatives, methods for their preparation, pharmaceutical compositioncontaining them and use thereof in treating certain mammalian tumors.The invention also relates to their use in the preparation ofconjugates.

Camptothecin and some of its analogs are cytotoxics that display potentantitumor activity by inhibiting topoisomerase I which is a monomericenzyme which controls the changes in DNA structure by catalyzing thebreaking and rejoining of the phosphodiester backbone of DNA strandsduring the normal cell cycle and it is thus involved in some importantcellular functions and cell growth. For a general reference tocamptothecin and its derivatives see for instance, Wani et al., J. Med.Chem: 1987, 30, 1774; Hsiang et al., Cancer Res. 1989, 49, 4385 andCancer Res. 1989, 49, 1465. Specifically, the synthesis and antitumoractivity of the 9-bromo-camptothecin is described in Chem Pharm Bull.1991, 39(12), 3183-3188.

Human DNA topoisomerase I (Topo I) is an essential enzyme that relaxesDNA supercoiling during replication and transcription and is recognizeto be validated target for the treatment of human cancers.

In recent years, Topo I has become a popular target for cancerchemotherapy treatments. It is thought that Topo I inhibitors such ascamptothecin and its analogs, block the ligation step of the cell cycle,generating single and double stranded breaks that harm the integrity ofthe genome. Introduction of these breaks subsequently lead to apoptosisand cell death (for a review on topoisomerase inhibitors see e.g.Oncogene 2003, 22, 7296-7304).

Topotecan and irinotecan are two camptothecin derivatives that areapproved for the treatment of a variety of malignancies, includingcolorectal, ovarian, and small cell lung cancers, as well as myeloidmalignancies.

There are, however, certain limitations to the use of the camptothecinderivatives in clinical. These include: low solubility, spontaneousinactivation to a lactone form in blood, rapid reversal of the trappedcleavable complex after drug removal, inadequate accumulation in thetumor, prolonged time of infusions, resistance of cancer cellsoverexpressing membrane transporters and dose-limiting side effects suchas diarrhea and neutropenia.

In addition, due to the low solubility, camptothecin required to beformulated as the ring-opened carboxylate salt that, other than beinginactive, is also considered responsible in part of its toxicity (see,for instance, Cancer Chemother. Rep, Part 1 1970, 54, 461; J. Pharm.Sci. 1992, 81, 676; Bioorg. Med. Chem. Lett. 2002, 12, 217).

Thus there is an increasing need of new camptothecin derivatives withhigh solubility so that to improve formulation and/orpharmacokinetic/pharmacodynamic properties.

Drug conjugation of cytotoxic drugs to molecules able to vehicle thedrug thus improving tumor targeting or able to modify itspharmacokinetic properties is one of the strategies that has beenundertaken to solve the above mentioned issues.

Different examples of conjugation of cytotoxics drugs with proteins,peptides, aptamers, polymers or nanoparticles allowing better targetdelivery, improving solubility and in some cases other pharmacokineticproperties such as increasing half life or local concentration of thedrug and improving drug performances have been reported. As a matter offacts, the resultant conjugates have improved characteristics in termsof solubility, permeability into the cell, in vivo therapeutic window,controlled release, ability to reach the target according to the natureo

molecule conjugated with the cytotoxic agent, etc.

For this reason, there is an increasing demand for the development offunctionalized cytotoxic agents suitable to be conjugated with differenttypes of molecules.

The first object of the present invention is to provide functionalized9-bromo-camptothecin derivatives which, other than having cytotoxicactivity, have high solubility so that to improve formulation and/orpharmacokinetic/pharmacodynamic properties. Furthermore thesefunctionalized 9-bromo-camptothecin derivatives are also suitable to beconjugated.

Accordingly, a first object of the present invention is to provide acompound of formula (I)

wherein:L is null or a conditionally-cleavable moiety;W is null or a self-immolative system, comprising one or moreself-immolative groups;Z is null or a peptidic, non peptidic or hybrid-peptidic and nonpeptidic-linker;RM is null or a reactive moiety that can be attached to one or more ofL, W or Z groups, provided that at least one ofL, W, Z and RM is not null, or RM is attached to oxygen when L, W and Zare all nullor a pharmaceutically acceptable salt thereof.

The present invention also provides methods of synthesizing thecompounds of formula (I), prepared through a process consisting ofstandard synthetic transformations, and isomers, tautomers, hydrates,solvates, complexes, metabolites, prodrugs, carriers and N-oxidesthereof.

The present invention also provides a method for treating cancer, whichcomprises administering to a mammal in need thereof an effective amountof a compound of formula (I) as defined above. The mammal in needthereof may be for example a human.

The present invention also provides a compound of formula (I), asdefined above, for use in a method of treating cancer, cellularproliferation disorders and viral infections.

Preferably, a compound of formula (I), as defined above, is for use in amethod of treating specific types of cancers, including but not limitedto: carcinomas, such as bladder, breast, colon, kidney, liver, lung,including small cell lung cancer, esophagus, gall-bladder, ovary,pancreas, stomach, cervix, thyroid, prostate, and skin, includingsquamous cell carcinoma; hematopoietic tumors of lymphoid lineage,including leukemia, acute lymphocitic leukemia, acute lymphoblasticleukemia, B-cell lymphoma, T-cell-lymphoma, Hodgkin's lymphoma,non-Hodgkin's lymphoma, hairy cell lymphoma and Burkitt's lymphoma;hematopoietic tumors of myeloid lineage, including acute and chronicmyelogenous leukemia, myelodysplastic syndrome and promyelocyticleukemia; tumors of mesenchymal origin, including fibrosarcoma andrhabdomyosarcoma; tumors of the central and peripheral nervous system,including astrocytoma, neuroblastoma, glioma and schwannoma; othertumors, including melanoma, teratocarcinoma, osteosarcoma, xerodermapigmentosum, keratoxanthoma, thyroid follicular cancer, Kaposi's sarcomaand mesothelioma.

Furthermore, a compound of formula (I), as defined above, is for use ina method of treating specific cellular proliferation disorders such as,for example, benign prostate hyperplasia, familial adenomatosispolyposis (FAP), neurofibromatosis, psoriasis, vascular smooth cellproliferation associated with atherosclerosis, pulmonary fibrosis,arthritis, glomerulonephritis and post-surgical stenosis and restenosis.

In addition, a compound of formula (I), as defined above is for use in amethod of inhibiting tumor angiogenesis and metastasis, as well as in amethod of treating organ transplant rejection and host versus graftdisease.

The present invention also provides a pharmaceutical compositioncomprising a therapeutically effective amount of a compound of formula(I), or a pharmaceutically acceptable salt thereof as defined above, andat least one pharmaceutically acceptable excipient, carrier or diluent.

The present invention further provides a pharmaceutical compositioncomprising a compound of formula (I), or a pharmaceutically acceptablesalt thereof as defined above, and one or more chemotherapeutic agents.

The present invention further provides a pharmaceutical compositioncomprising a compound of formula (I), or a pharmaceutically acceptablesalt thereof as defined above, in combination with known anticancertreatments, such as radiation therapy or chemotherapy regimen, and/or incombination with cytostatic or cytotoxic agents, antibiotic-type agents,alkylating agents, antimetabolite agents, hormonal agents, immunologicalagents, interferon-type agents, cyclooxygenase inhibitors (e.g. COX-2inhibitors), matrixmetalloproteinease inhibitors, telomerase inhibitors,tyrosine kinase inhibitors, anti-growth factor receptor agents,anti-HER2 agents, anti-EGFR agents, anti-angiogenesis agents (e.g.angiogenesis inhibitors), farnesyl transferase inhibitors, ras-rafsignal transduction pathway inhibitors, cell cycle inhibitors, othercdks inhibitors, tubulin binding agents, topoisomerase I inhibitors,topoisomerase II inhibitors, and the like.

Additionally, the invention provides a product comprising a compound offormula (I), or a pharmaceutically acceptable salt thereof as definedabove, and one or more chemotherapeutic agents, as a combinedpreparation for simultaneous, separate or sequential use in anticancertherapy.

In yet another aspect the invention provides a compound of formula (I)or a pharmaceutically acceptable salt thereof, as defined above, for useas a medicament.

Moreover the invention provides the use of a compound of formula (I), ora pharmaceutically acceptable salt thereof as defined above, in themanufacture of a medicament with anticancer activity.

Finally, the invention provides the use of a compound of formula (I) ora pharmaceutically acceptable salt thereof, as defined above, in thepreparation of conjugates.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the deconvoluted mass spectrum of unreacted MCM2 proteinand reports the molecular weight (m/z) on the x axis, while intensityexpressed as counts per second (cps) is reported on the y axis.

FIG. 2 shows the deconvoluted mass spectrum of MCM2 protein reacted withComp. 8 and reports the molecular weight (m/z) on the x axis, whileintensity expressed as counts per second (cps) is reported on the yaxis.

FIG. 3 shows the HPLC profile of the conjugate A1 and reports the time(min) on the x axis while UV (mAU) is reported on the y axis.

FIG. 3 a shows the mass spectrum of the conjugate A1 and reports themolecular weight (m/z) on the x axis while intensity expressed as countsper second (cps) is reported on the y axis.

FIG. 4 shows the HPLC profile after 2 h treatment of conjugate A1 withcathepsin and reports the time (min) on the x axis while UV absorbance(mAU) is reported on the y axis.

FIG. 4 a shows the mass spectrum of the released Comp. 11 and reportsthe molecular weight (m/z) on the x axis while intensity expressed ascounts per second (cps) is reported on the y axis.

FIG. 5 shows the HPLC profile of Comp. 11 and reports the time (min) onthe x axis while UV absorbance (AU) is reported on the y axis.

FIG. 5 a shows the HPLC profile after 1 h treatment at pH 7.4 of Comp.11 and reports the time (min) on the x axis while UV absorbance (AU) isreported on the y axis.

FIG. 5 b shows the mass spectrum of the released 9-bromo-camptothecinand reports the molecular weight (m/z) on the x axis while intensityexpressed as counts per second (cps) is reported on the y axis.

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings.

With the term “linear or branched C₁-C₄ alkyl” we intend any of thegroups such as, for instance, methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl.

With the term “linear or branched C₁-C₄ hydroxyalkyl” we intend any ofthe groups such as, for instance, 2-hydroxyethyl, 3-hydroxypropyl,2-hydroxypropyl, 4-hydroxybutyl, 3-hydroxybutyl, 2-hydroxybutyl.

With the term “linear or branched C₁-C₄ alkoxy”, we intend any of thegroups such as, for instance, methoxy, ethoxy, propoxy, tert-butoxy,etc.

The term “polyfluorinated alkyl” or “polyfluorinated alkoxy” means anyof the above linear or branched C₁-C₄ alkyl or alkoxy groups which aresubstituted by more than one fluorine atom such as, for instance,trifluoromethyl, trifluoroethyl, 1,1,1,3,3,3-hexafluoropropyl,trifluoromethoxy and the like.

With the term “halogen” we intend a fluorine, chlorine, bromine oriodine.

With the term “linear or branched C₁-C₄ aminoalkyl” we intend any of thegroups such as, for instance, 2-aminoethyl, 3-aminopropyl, 4-aminobutyl,3-aminobutyl, etc.

The term “linear or branched C₁-C₄ sulfhydrylalkyl” as used hereinrefers to a —SH group appended to a linear or branched C₁-C₄ alkylgroup, as previously defined.

The term “C₃-C₈ cycloalkyl” as used herein refers to a saturated orunsaturated non-aromatic all-carbon monocyclic ring, which may consistof one ring or two or more rings fused together. Examples include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl,cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl,decalinyl, and 1,4-cyclohexadienyl.

The term “heterocyclyl” as used herein refers to a saturated orunsaturated non-aromatic C₄-C₈ carbocyclic ring which may consist of onering or two or more rings fused together, wherein from 1 to 4 carbonatoms are replaced by heteroatoms such as nitrogen, oxygen, sulfur,wherein said heteroatoms may be directly connected to each other;nitrogen and sulfur may optionally be oxidized and nitrogen mayoptionally be quaternized. Non limiting examples of heterocyclyl groupsare, for instance, tetrahydrofuranyl, pyrrolidinyl, piperidinyl,1,4-dioxanyl, decahy

piperazinyl, oxazolidinyl and morpholinyl.

The term “aryl” as used herein refers to a mono-, bi- orpoly-carbocyclic hydrocarbon from 1 to 4 ring systems, optionallyfurther fused or linked to each other by single bonds, wherein at leastone of the carbocyclic rings is aromatic, wherein the term “aromatic”refers to a completely conjugated 7-electron bond system. Non limitingexamples of such aryl groups are phenyl, α- or β-naphthyl or anthracenylgroups.

The term “heteroaryl” as used herein refers to aromatic heterocyclicrings, typically 4- to 7-membered heterocycles, with from 1 to 4heteroatoms selected among oxygen, nitrogen and sulfur, wherein nitrogenand sulfur may optionally be oxidized and nitrogen may optionally bequaternized; said heteroaryl ring can be optionally further fused orlinked to one or two or more rings fused together, aromatic andnon-aromatic carbocyclic and heterocyclic rings. Heteroatoms may bedirectly connected to each other. Examples of heteroaryl groups include,but are not limited to, pyridyl, pyrimidyl, furanyl, pyrrolyl,triazolyl, pyrazolyl, pyrazinyl, oxazolyl, isoxazolyl, thiazolyl,imidazolyl, thienyl, indolyl, benzofuranyl, benzimidazolyl,benzothiazolyl, purinyl, indazolyl, benzotriazolyl, benzisoxazolyl,quinoxalinyl, isoquinolyl, and quinolyl. In one embodiment, a heteroarylgroup comprises from 1 to 4 heteroatoms. It should be noted that “C₁heteroaryl group” denotes that there is only one carbon present in thering system of the heteroaromatic group (carbon atoms in optionalsubstituents are thus not counted). An example of such a heteroaromaticgroup is tetrazolyl group.

The term “activating moiety” refers to a functional group that improveschemical reactivity of the chemical function to which the activatingmoiety is attached. Chemical functions that can be activated in such wayare, for instance, amine and carbonyl. Examples of activated amines arealkylsulfonyloxyamine, alkylsulfonyloxycarbamate,phenylsulfonyloxyamine, phenylsulfonyloxycarbamate. Examples ofactivated carbonyl groups are, for example, activated esters.

The term “activated ester” refers to a functional group in which thealkoxy group of the ester moiety is a good leaving group. Examples ofsuch alkoxy groups include, but are not limited to, succinimide-N-oxide,p-nitrophenoxide, pentafluorophenoxide, tetrafluorophenoxide,1-hydroxybenzotriazole, and 1-hydroxy-7-azabenzotriazole, and groupswith comparable leaving capability. Unsubstituted alkyl-based alkoxygroups such as methoxy, ethoxy, isopropoxy, and tert-butoxy do notqualify as good leaving groups and methyl, ethyl, isopropyl, andtert-butyl esters are therefore not considered to be activated esters.

It is known to the person skilled in the art that transformation of achemical functional group into another may require that one or morereactive centers in the compound containing such functional group haveto be protected in order to avoid undesired side reactions. Protectionof such reactive centers, and subsequent deprotection at the end of thesynthetic transformations, can be accomplished following standardprocedures described in the literature (see, for instance, Green,Theodora W. and Wuts, Peter G. M.—Protective Groups in OrganicSynthesis, Third Edition, John Wiley & Sons Inc., New York (N.Y.),1999).

Therefore, the term “protecting group” refers to a group used to protectsuch reactive centers in a chemical synthesis, for example, a hydroxylgroup (—OH), an amino group (—NH), a thiol group (—SH), a carbonyl group(—C═O), a carboxylic group (—COOH). Examples of protecting groups arethose reported in the literature (see, for instance, ibidem).

The term “nucleophiles” refers to molecules that bear a nucleophilicgroup. The term “nucleophilic gr

species that donates an electron-pair to an electrophilic group to forma chemical bond in a chemical reaction. Examples of such nucleophilicgroups include, but are not limited to halogens, amines, nitrites,azides, alcohols, alkoxyde anions, carboxylate anions, thiols,thiolates, etc.

The term “electrophilic group” refers to a species that accepts anelectron-pair from a nucleophilic group to form a chemical bond in achemical reaction. Examples of such electrophilic groups include, butare not limited to esters, aldehydes, amides, ketons, etc.

The term “unnatural aminoacid” refers to the D-stereoisomer of thenaturally occurring aminoacid (L-stereoisomer). Pharmaceuticallyacceptable salts of the compounds of formula (I) include the acidaddition salts with inorganic or organic acids, e.g., nitric,hydrochloric, hydrobromic, sulfuric, perchloric, phosphoric, acetic,trifluoroacetic, propionic, glycolic, fumaric, lactic, oxalic, malonic,malic, maleic, tartaric, citric, benzoic, cinnamic, mandelic,methanesulphonic, isethionic and salicylic acid.

Pharmaceutically acceptable salts of the compounds of formula (I) alsoinclude the salts with inorganic or organic bases, e.g., alkali oralkaline-earth metals, especially sodium, potassium, calcium ammonium ormagnesium hydroxides, carbonates or bicarbonates, acyclic or cyclicamines.

If a stereogenic center or another form of an isomeric center is presentin a compound of the present invention, all forms of such isomer orisomers, including enantiomers and diastereoisomers, are intended to becovered herein. Compounds containing a stereogenic center may be used asa racemic mixture, an enantiomerically enriched mixture, or the racemicmixture may be separated using well-known techniques and an individualenantiomer may be used alone. In cases in which compounds haveunsaturated carbon-carbon double bonds, both the cis (Z) and trans (E)isomers are within the scope of this invention.

In cases when compounds can exist in tautomeric forms, each form iscontemplated as being included within this invention whether existing inequilibrium or predominantly in one form.

The Conditionally-Cleavable Moiety L

The L moiety, if present, is a conditionally-cleavable group that can becleaved by a chemical, photochemical, physical, biological or enzymaticprocess upon being brought in or under certain conditions. One of theseconditions may for example be bringing a compound of the invention in anaqueous environment, which leads to hydrolysis of L or bringing acompound of the invention in an environment that contains an enzyme thatrecognizes and cleaves L, or bringing a compound of the invention underreducing conditions, which leads to reduction and/or removal of L, orbringing a compound of the invention under oxidizing conditions, whichleads to oxidation and removal of L, or bringing a compound of theinvention in contact with radiation, e.g., UV light, which leads tocleavage, or bringing a compound of the invention in contact with heat,which leads cleavage of L. This condition may be met directly afteradministering a compound of this invention to an animal, e.g. a mammal,for example a human, due to the presence of ubiquitous enzymes in thecirculation. Alternatively, said condition may be met when the compoundlocalizes to a specific organ, tissue, cell, subcellular target, orbacterial, viral, or microbial target, for example by the presence ofinternal factors (e.g. target-specific enzymes or hypoxia) orapplication of external factors (e.g. radiation, magnetic fields).

Cleavage of L means that the bond between the oxygen and L in a compoundof formula (I) is broken:

In one embodiment, L can be a moiety that is cleaved by an enzyme orhydrolytic conditions present in the vicinity or inside the target cellsas compared to other parts of the body, or by an enzyme or hydrolyticconditions present only in the vicinity of or inside the target cells.It is important to recognize that if target site specificity is achievedsolely based upon the selective transformation and/or cleavage of said Lat the target site, the condition causing the cleavage shouldpreferably, at least to a certain degree, be target site-specific.

In one embodiment, cleavage of L occurs intracellularly.

In another embodiment, cleavage of L occurs extracellularly.

In another embodiment, cleavage of L can occur by a ubiquitousintracellular enzyme.

In one preferred embodiment, L may be a moiety that can be cleaved byubiquitous enzymes, e.g., esterases that are present in the circulationor intracellular enzymes, such as for example proteases andphosphatases, or by pH-controlled hydrolysis. L may therefore form,optionally together with the connecting atom oxygen, a hydroxylamine,carbamate, urea, ester, ether, acetal, ketal or phosphate group that canbe cleaved in vivo.

In a more preferred embodiment L is independently null or a groupselected from:

wherein:R1, R2 and R3 are, each independently, null, hydrogen, hydroxy or anoptionally substituted group selected from linear or branched C₁-C₄alkyl, linear or branched C₁-C₄ hydroxyalkyl, linear or branched C₁-C₄sulfhydrylalkyl, linear or branched C₁-C₄ aminoalkyl, linear or branchedC₁-C₄ alkylcarbonyl and linear or branched C₁-C₄ alkoxycarbonyl;each of n is independently an integer from 0 to 2, andn1 is an integer from 0 to 5.* indicates the attachment points to the other moieties in the compoundof formula (I), herewith and throughout the application.

According to the present invention and unless otherwise provided, theabove R1, R2 and R3 groups may be optionally substituted, in any oftheir free positions, by one or more groups, for instance 1 to 3 groups,independently selected from: halogen, linear or branched C₁-C₄ alkyl,polyfluorinated C₁-C₄ alkyl, linear or branched C₁-C₄ alkoxy,polyfluorinated C₁-C₄ alkoxy, hydroxy, amino, linear or branched C₁-C₄alkylamino, di-C₁-C₄ alkyl,

alkylcarbonyl, C₃-C₈ cycloalkyl, C₃-C₈ cycloalkyl-C₁-C₄ alkyl,heterocyclyl, heterocyclyl-C₁-C₄ alkyl, aryl, aryl-C₁-C₄ alkyl,heteroaryl and heteroaryl-C₁-C₄ alkyl.

The Self-Immolative System W

The W group, if present, is a self-immolative system that in a compoundof formula (I) tethers in a stable way a moiety L (or oxygen, if L isnull) to a moiety Z (or RM, if Z is null). The L-W, or O-W, bond canbecome labile upon activation by a chemical, photochemical, physical,biological or enzymatic process upon being brought in or under certainconditions, as described above, leading optionally to the release of thecorresponding moieties:

A self-immolative system may be incorporated in a compound of formula(I) for example to improve solubility or to improve space between the9-bromo-camptothecin derivative and the reactive moiety; in additionsaid self-immolative group can modulate the reactivity of RM versusnucleophiles.

Self-immolative systems are known to the person skilled in the art, seefor example those described in WO2002/083180 and WO2004/043493; or thosedescribed in Tranoy-Opalinsi, A. et al., Anticancer Agents in MedicinalChemistry, 2008, 8, 618-637. Other examples of self-immolative groupsinclude, but are not limited to, optionally substituted 4-aminobutyricacid amides, appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2]ring systems or 2-aminophenylpropionic acid amides [see WO 2005/079398,WO 2005/105154 and WO 2006/012527; Greenwald, R. B., et al., Adv. DrugDelivery Rev. 2003, 55, 217-250; Kingsbury, W. D., et al., J. Med. Chem.1984, 27, 1447-1451].

In one preferred embodiment, W may form, together with the connectingatom(s) oxygen, L, Z, or RM, a carbonate, carbamate, urea, ester,thioamide, phosphate, amide, hydroxylamine or ether linkage that can beoptionally cleaved upon activation.

In a more preferred embodiment, W is independently null or a groupselected from:

whereinone of R1 and R2 is null and the other is as defined above;R3 is as defined above;R4 and R5 are, each independently, halogen, methyl, ethyl or linear orbranched C₁-C₄ hydroxyalkyl;m is an integer from 0 to 3;A is C₁-C₃ alkyl, CH₂NH, NH or N—R4, wherein R4 is as defined above; andR12 and R13 are, each independently, hydrogen, halogen, methyl, ethyl,linear or branched C₁-C₄ hydroxyalkyl, linear or branched C₁-C₄haloalkyl, or R12 and R13 taken together form a 3- to 6-memberedcarbocycle.

In another more preferred embodiment, W is independently null or a groupselected from:

wherein one of R1 and R2 is null and the other is as defined above; andR4, R5 and A are as defined above.

The Z Linker

The Z linker, if present, can be peptidic (Z1), non-peptidic (Z2) orhybrid (Z3), wherein said hybrid linker is peptidic and non-peptidic; ina compound of formula (I) said Z linker can be cleaved from theleft-hand side moiety (W, L or oxygen) by a chemical, photochemical,physical, biological or enzymatic process upon being brought in or undercertain conditions, as described above:

The Z linker may be linear or branched.

The linkage between Z and its left-hand side moiety or between Z and,optionally, RM may be an amide, a carbonate, an urea, an ester, athioamide, a disulfide, an hydroxylamine or a carbamate linkage.

In one embodiment Z is null.

In another embodiment Z is a peptidic linker Z1 that can be cleaved by aproteolytic enzyme, plasmin, a cathepsin, p-glucuronidase, agalactosidase, prostate-specific antigen (PSA), urokinase-typeplasminogen activator (u-PA) or a member of the family of matrixmetalloproteinases.

In another embodiment Z, is a non-peptidic linker Z2 that may containone or more non-peptidic water-soluble moieties. In this case the linkercontributes to the water solubility of a compound of formula (I). Inanother embodiment Z2 is a non-peptidic linker that may contain one ormore non-peptidic moieties that reduce(s) aggregation of a compound offormula (I) which may or may not be a moiety/moieties that alsoincrease(s) the water solubility of a compound of formula (I).

For example, non-peptidic water-soluble Z2 linkers may contain anoligoethylene glycol or polyethylene glycol moiety or a derivativethereof.

In another embodiment, Z is a hybrid linker Z3 that can contain bothpeptidic and non peptidic residu

formula Z1-Z2, wherein Z1 and Z2 are independently a peptidic linker ora non-peptidic linker. Hybrid linkers may contribute to solubility ofthe compound of formula (I) and/or may be a substrate that can becleaved by proteolytic enzymes, for example by a member of the family ofmatrix metalloproteinases.

In a preferred embodiment, Z1 is a single aminoacid, a dipeptide, atripeptide, a tetrapeptide, or an oligopeptide moiety comprising naturalL-aminoacids, unnatural D-aminoacids, synthetic aminoacids, or anycombination thereof, wherein one of the C-terminal or the N-terminalaminoacid residue is linked to W, L or oxygen, and the other terminalaminoacid ends with a COOH or NH₂ group or is optionally linked to RM.

In a more preferred embodiment Z1 is a dipeptide or a tripeptide, linkedvia its C-terminus to W, or to L when W is null, or to oxygen when W andL are both null.

In another more preferred embodiment, the C-terminal aminoacid residueof the dipeptide or of the tripeptide is selected from glycine, leucine,alanine, arginine and citrulline; and the N-terminal aminoacid residueis selected from any natural or unnatural aminoacid; preferably, in caseof the tripeptide, the middle aminoacid residue is selected fromalanine, valine, leucine, isoleucine, methionine, phenylalanine andproline.

In another more preferred embodiment Z1 comprises a pentapeptide,wherein the C-terminal aminoacid is selected from any natural orunnatural aminoacid and the N-terminal aminoacid residue is6-aminohexanoic acid.

In a preferred embodiment, Z2 may contain an oligoethylene glycol orpolyethylene glycol moiety or a derivative thereof.

In a more preferred embodiment Z2 is a group selected from:

whereinone of R1 and R2 is null and the other is as defined above andp is an integer from 1 to 20.

In a preferred embodiment Z3 is a hybrid moiety comprising a peptidicmoiety Z1, wherein Z1 is a single aminoacid, a tripeptide or atetrapeptide, comprising natural L-aminoacids and unnaturalD-aminoacids; and a non-peptidic moiety Z2, wherein Z2 is anoligoethylene glycol or polyethylene glycol moiety or a derivativethereof.

In another preferred embodiment Z is a group selected from:

whereinone of R1 and R2 is null and the other is as defined above.

The Reactive Moiety RM

The RM moiety, if present, is an electrophilic group that can react withnucleophiles, i.e. molecules that bear a nucleophilic group, underrelatively mild conditions and without the need of priorfunctionalization of the reactive moiety, said reaction between saidreactive moiety and said nucleophile requiring only the application ofone or more of the agents selected from the group comprising heat,pressure, a catalyst, an acid and a base.

Therefore, when the RM moiety is present, a compound of formula (I)conjugates with different types of nucleophiles. When RM is null, acompound of formula (I) conjugates with different types ofelectrophiles, i.e. molecules that bear an electrophilic group, throughone or more of the nucleophilic groups that are present on the L, W,and/or Z moiety(ies).

In a compound of formula (I) the RM moiety can be connected to one ormore of the L, W or Z groups or to the oxygen atom of the9-bromo-camptothecin derivative:

Examples of reactive moieties include, but are not limited to, carbamoylhalide, acyl halide, activated ester, anhydride, α-haloacetyl,α-haloacetamide, maleimide, isocyanate, isothiocyanate, disulfide,thiol, hydrazine, hydrazide, sulfonyl chloride, aldehyde, methyl ketone,vinyl sulfone, halomethyl, and methyl sulfonate.

In one preferred embodiment of the invention, when the nucleophilicgroup of the nucleophile, which RM can react with, is NH, NH₂, SH or OH,RM is independently null or a group selected from

wherein R6 is C₁-C₃ alkyl or an electron-withdrawing group, comprisingNO₂ and CN group;r is an integer from 0 to 7, andR4 and R5 are as defined above.

In another preferred embodiment of the invention, when the nucleophilicgroup of the nucleophile, which RM can react with, is COOH, RM isindependently null or a group selected from

Specific, not limiting, preferred compounds (Comp.) of the presentinvention, optionally in the form of a pharmaceutically acceptable salt,are the following:

-   1)    (4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl    glycinate;-   2)    (4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl    L-phenylalanyl-L-leucylglycinate;-   3)    (4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl    piperazine-1-carboxylate;-   4)    (4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl    N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-L-leucylglycinate;-   5)    N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[4-({[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}carbonyl)piperazin-1-yl]carbonyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   6)    (4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl    4-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]piperazine-1-carboxylate;-   7)    (4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl    methyl[2-(methylamino)ethyl]carbamate;-   8)    N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[{2-[({[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide;-   9)    (4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quir    amino-3,3-dimethylbutanoate hydrochloride;-   10)    (4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl    5-amino-4,4-difluoropentanoate hydrochloride;-   11)    4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl    [1-(aminomethyl)cyclohexyl]acetate hydrochloride;-   12)    L-valyl-N-[4-({[(5-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-difluoro-5-oxopentyl)carbamoyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide;-   13)    L-valyl-N-[4-({[(4-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-dimethyl-4-oxobutyl)carbamoyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide;-   14)    L-valyl-N-(4-{[({[1-(2-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2-oxoethyl)cyclohexyl]methyl}carbamoyl)oxy]methyl}phenyl)-N⁵-carbamoyl-L-ornithinamide;-   15)    N-[6-(2,5-dioxopyrrolidin-1-yl)hexanoyl]-valyl-N-[4-({[(5-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-difluoro-5-oxopentyl)carbamoyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide;-   16)    N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-(5-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-difluoro-5-oxopentyl)-N⁵-carbamoyl-L-ornithinamide;-   17)    N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{[1-(2-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2-oxoethyl)cyclohexyl]methyl}-N⁵-carbamoyl-L-ornithinamide;-   18)    N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-(4-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-dimethyl-4-oxobutyl)-N⁵-carbamoyl-L-ornithinamide;-   19)    N-(3-carboxypropanoyl)-L-valyl-N-(5-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-difluoro-5-oxopentyl)-N⁵-carbamoyl-L-ornithinamide;-   20)    N-(3-carboxypropanoyl)-L-valyl-N-{[1-(2-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2-oxoethyl)cyclohexyl]methyl}-N⁵-carbamoyl-L-ornithinamide;-   21)    N-(3-carboxypropanoyl)-L-valyl-N-(4-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-dimethyl-4-oxobutyl)-N⁵-carbamoyl-L-ornithinamide;-   22)    N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-N-(5-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-difluoro-5-oxopentyl)-L-leucinamide;-   23)    N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-N-{[1-(2-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2-oxoethyl)cyclohexyl]methyl}-L-leucinamide;-   24)    N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-N-(4-{[(4S)-10-bromo-4-et    3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-dimethyl-4-oxobutyl)-L-leucinamide;-   25)    L-valyl-N-{[1-(2-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2-oxoethyl)cyclohexyl]methyl}-N⁵-carbamoyl-L-ornithinamide;-   26)    L-valyl-N-(4-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-dimethyl-4-oxobutyl)-N⁵-carbamoyl-L-ornithinamide,    and-   27)    L-valyl-N-(5-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-difluoro-5-oxopentyl)-N⁵-carbamoyl-L-ornithinamide.

For a reference to any specific compound of formula (I) of theinvention, optionally in the form of a pharmaceutically acceptable salt,see the experimental section and claims.

The present invention also provides a process for the preparation of acompound of formula (I), as defined above, by using the reaction routesand synthetic schemes described below, employing the techniquesavailable in the art and starting materials readily available. Thepreparation of certain embodiments of the present invention is describedin the examples that follow, but those of ordinary skill in the art willrecognize that the preparations described may be readily adapted toprepare other embodiments of the present invention. For example, thesynthesis of non-exemplified compounds according to the invention may beperformed by modifications apparent to those skilled in the art, forinstance by appropriately protecting interfering groups, by employingother suitable reagents known in the art, or by making routinemodifications of reaction conditions. Alternatively, other reactionsreferred to herein or known in the art will be recognized as havingadaptability for preparing other compounds of the invention.

All those with ordinary skills in the art will appreciate that anytransformation performed according to said methods may require standardmodifications such as, for instance, protection of interfering groups,change to other suitable reagents known in the art, or make routinemodifications of reaction conditions.

Accordingly, the process for the preparation of a compound of formula(I) is depicted in Scheme 1 below:

The present invention provides a process for the preparation of acompound of formula (I) as defined above, characterized in that theprocess comprises the following steps:

1) reacting a compound of formula (VI)

wherein R7 is hydrogen, alternatively with:

-   -   1a) a compound of formula (VIIa)

R8-L  (VIIa)

-   -   wherein R8 is an activating moiety of the NH group, preferably        tosyl, and L is a group of formula —NHCOR1 (IIa), —NHCONH—R1        (IIb), —NHCOO—R1 (IIc), or —NH—R1 (IId), wherein R1 is as        defined above but not null, and then removing the protecting        group if present;    -   or    -   1b) a compound of formula (VIIb)

Q-O-L  (VIIb)

-   -   wherein Q is hydrogen or an activating moiety of the carboxylic        group, e.g. an activated ester, and L is a group of formula        (IIe) or (IIk)

-   -   wherein    -   R1 and R2 are as defined above but not null and n1 is as defined        above, and then removing the protecting group if present;    -   or    -   1c) a compound of formula (VIIf) or (VIIg)

-   -   wherein n is as defined above ad R1 is as defined above but not        null, and then removing the protecting group if present;    -   or    -   1d) a compound of formula (VIIh) or (VIIi)

-   -   wherein R1 is as defined above but not null and n is as defined        above, and then removing        group if present;    -   or    -   1e) reacting a compound of formula (VI) as defined above,        wherein R7 is an imidazol-1-yl-carbonyl moiety, with a compound        of formula (VIIj) or (VIIm)

-   -   wherein R1 is as defined above but not null, and R2, R3 and n1        are as defined above;        2) converting the resultant compound of formula (Ia)

wherein L is a group of formula (IIa) to (IIm), wherein R1 is hydrogenor hydroxy, and R2, R3, n and n1 are as defined above, alternativelyinto:

-   -   2a) a compound of formula (I) as defined above, by reaction with        a compound of formula (VIII)

-   -   wherein R9 is hydrogen, hydroxy or a carbonyl group activating        moiety, preferably 4-nitrophenyl-oxy, and W, Z and RM are as        defined above but not all null, and then removing the protecting        group if present;    -   or into    -   2b) a compound of formula of formula (Ib)

-   -   wherein L and W are as defined above, and Z is a group Z1,        wherein the ending aminoacid has a COOH or NH free group; or a        group Z2 of formula (IVa)-(IVp), wherein R1 or R2 is selected        from hydrogen and hydroxy; or a group Z3, wherein Z1 and Z2 are        as defined above, by reaction with a compound of formula (IX)

R9-W-Z  (IX)

-   -   wherein R9 and W are as defined above, and Z is a group Z1,        wherein the ending aminoacid or NH free group; or a group Z2 of        formula (IVa)-(IVp), wherein R1 or R2 is selected from hydrogen        and hydroxy; or a group Z3, wherein Z1 and Z2 are as defined        above, and then removing the protecting group if present;        then        3) converting a compound of formula (Ib) as defined above, to a        compound of formula (I) as defined above, by reaction with a        compound of formula (X)

R10-RM  (X)

wherein R10 is halogen, hydroxy, or an activating moiety of thecarboxylic group, e.g. an activated ester;or4) reacting a compound of formula (VI) as defined above, alternativelywith:

-   -   4a) a compound of formula (XI)

-   -   wherein R11 is a group of formula (VIIa), (VIIb), (VIIf),        (VIIg), (VIIh) or (VIIi), wherein R1 is null, to obtain a        compound of formula (I) wherein W, Z and RM are as defined above        and L is as defined above but not null;    -   or with    -   4b) a compound of formula (VIII) as defined above,    -   to obtain a compound of formula (I) as defined above, wherein L        is null and W, Z and RM are as defined above,        or a pharmaceutically acceptable salt thereof.

Alternatively, a compound of formula (I) can be also prepared accordingto Scheme 2 depicted below:

characterized in that the process comprises the following steps:5) reacting a compound of formula (VI) as defined above, with a compoundof formula (XII)

R9-W  (XII)

wherein R9 is as defined above, and W is as defined above, wherein R1and R2 are as defined above but not null, and then removing theprotecting group if present;then, reacting the resultant compound of formula (Ic)

wherein W is a group (IIIa)-(IIIe), wherein R1 and R2 are as definedabove but not null, alternatively with,

-   -   6a) a compound of formula (XIII)

-   -   wherein R9 is as defined above and Z and RM are as defined above        but not all null, to obtain a compound of formula (I) wherein W        is as defined above but not null, and Z and RM are as defined        above but not null; or with    -   6b) a compound of formula (XIV)

R9-Z  (XIV)

-   -   wherein R9 is as defined above and Z is a group Z1, wherein the        ending aminoacid has a COOH or NH free group; or a group Z2 of        formula (IVa)-(IVp), wherein R1 or R2 is selected from hydrogen        and hydroxy; or a group Z3, wherein Z1 and Z2 are as defined        above, and then removing the protecting group if present; and    -   7) finally, reacting the resultant compound of formula (Id)

-   -   wherein W is as defined above but not null and Z is a group Z1,        wherein the ending aminoacid has a COOH or NH free group; or a        group Z2 of formula (IVa)-(IVp), wherein R1 or R2 is selected        from hydrogen and hydroxy; or a group Z3, wherein Z1 and Z2 are        as defined above, with a compound of formula (X)

R10-RM  (X)

-   -   wherein R10 is as defined above, to obtain a compound of formula        (I), wherein W is as defined above but not null    -   and Z and RM are as defined above but RM is not null;        or        8) reacting a compound of formula (VI) as defined above with:    -   8a) a compound of formula (VIII) as defined above, to obtain a        compound of formula (I), wherein W, Z and RM are as defined        above but not all null,    -   or with    -   8b) a compound of formula (XIII) as defined above, to obtain a        compound of formula (I), wherein W is null, and Z and RM are as        defined above but not all null;        or a pharmaceutically acceptable salt thereof.

According to step 1a) the reaction is performed in an organic solvent,preferably diethyl ether, dioxane or a mixture thereof, with LiHMDS at atemperature ranging from −10° C. to 50° C. and for a time ranging from30 min to about 24 h (J. Med. Chem., 2010, 53, 7731).

According to step 1b) the reaction is performed in an organic solvent,preferably DMSO, DCM, THF, CH₃CN or CCl₄, optionally in the presence ofa base, preferably DIPEA or DMAP, at a temperature ranging from −10° C.to 50° C. and for a time ranging from 30 min to about 5 days.

According to step 1c) and 1d) the reaction is carried out in an organicsolvent, preferably DCM or DMF, in the presence of PTSA at a temperatureranging from 20° C. to reflux and for a time ranging from 30 min toabout 24 h.

According to step 1e) the coupling reaction is performed in an organicsolvent, preferably DCM or CH₃CN, in optionally basic conditions, e.g.TEA, DMAP, and optionally in presence of a condensing agent such as DCCor EDCl. The reaction is carried out at a temperature ranging from 0° C.to reflux and for a time ranging from 30 min to about 24 h. Removal ofthe protecting group is performed using known procedure reported in theliterature (see e.g. Protective Groups in Organic Synthesis; Theodora W.Greeen, Peter G. M. Wuts).

According to step 2a), 2b), 3), 5), 6a), 6b) and 7) the reaction isperformed in an organic solvent, preferably DCM, THF, DMSO or DMF inoptionally basic conditions, e.g. TEA, DMAP and optionally in presenceof a condensing agent such as DCC, EDCl and optionally in presence of atriflate salt, preferably, scandium(III) triflate. The reaction iscarried out at a temperature ranging from 0° C. to reflux and for a timeranging from 30 min to about 24 h.

According to step 4a) the coupling reaction is performed as reportedabove under steps 1a) to 1e) depending upon the characteristics of thereagents applied.

According to step 4b) the coupling reaction is performed in an organicsolvent, preferably DMSO, DCM, THF, CH₃CN or DMF, optionally in presenceof a condensing agent, such as for example DCC, EDC and with theoptional addition of DMAP (for general coupling reagents see e.g. AminoAcids, Peptides and Proteins in Organic Chemistry: Building Blocks,Catalysis and Coupling Chemistry, Volume 3; Andrew B. Hughes, AymanEl-Faham, Fernando Albericio). The coupling reaction according to step4b) is preferably carried out at a temperature ranging from 20° C. toreflux, in the optional presence of a base, and for a time ranging from30 min to about 24 h.

The compound of formula (VI) wherein R7 is hydrogen can be prepared asreported in Chem. Pharm. Bull. 1991, 39(12), 3183-3188; the compound offormula (VI) wherein R7 is an imidazol-1-yl-carbonyl moiety is preparedas reported in the experimental part (see below).

Compounds of formula (VII) are commercially available or can be preparedby methods known to the expert in the art or as reported in WO9202255;J. Med. Chem. 2010, 53 (21), 7731-7738; J. Med. Chem., 2012, 55 (2),766-782.

Compounds of formula (VIII), (IX), (X), (XI), (XII), (XIII) and (XIV)are commercially available or can b

methods known to the expert in the art or as reported in AnticancerAgents in Med Chem 2008, (8), 618-637 or in WO2010/009124, EP0624377 orEP2357006.

Pharmacology

The compounds of the present invention are useful as antitumor agents.

A mammal, e.g. a human or animal, may therefore be treated by a methodcomprising administering thereto a pharmaceutically effective amount ofa compound of formula (I).

The condition of the human or animal may be ameliorated or improved inthis way.

The evaluation of the cytotoxicity of the compounds of formula (I) isassessed as described below.

In Vitro Cell Proliferation Assay

A2780 human ovarian and MCF7 human breast cancer cells (1250 cells/well)were seeded in white 384 well-plates in complete medium (RPMI1640 orEMEM plus 10% Fetal bovine serum) and treated with compounds dissolvedin 0.1% DMSO, 24 h after seeding. The cells were incubated at 37° C. and5% CO₂ and after 72 h the plates were processed using CellTiter-Gloassay (Promega) following the manufacturer's instructions.

CellTiter-Glo is a homogenous method based on the quantification of theATP present, an indicator of metabolically active cells. ATP isquantified using a system based on luciferase and D-luciferin resultinginto light generation. The luminescent signal is proportional to thenumber of cells present in culture.

Briefly, 25 μL/well of reagent solution are added to each well and after5 min shaking microplates are read by a luminometer. The luminescentsignal is proportional to the number of cells present in culture.

Representative compounds of the invention of formula (I) were tested inthe specific in vitro cell proliferation assay described above.

All the tested compounds have an IC₅₀ value <100 nM in A2780 humanovarian cancer cells.

In particular, Comp. 1 and 2 have IC₅₀ value <10 nM.

Compound A2780 (IC₅₀ nM) 9-bromo-camptothecin 1.625 1 0.970 2 1.559

As can be appreciated by the skilled person, all these representativecompounds are thus particularly advantageous in antitumor therapy.

Furthermore, the functionalized compounds of formula (I) of the presentinvention are suitable to be conjugated. The ability of thefunctionalized derivatives of formula (I) to be conjugated has beenassessed by conjugating them with the MCM2 protein.

Solubility Assay: High-Throughput Solubility

Nominal 200 μM compounds suspensions/solutions in aqueous potassiumphosphate buffer at pH=7 and aqueous citric acid buffer at pH=3 wereprepared on a “Multiscreen-HTS”, 0.2 μm filter plate (Millipore,Billerica, Mass.). These solutions were stirred for 10 min and storedfor 24 h at room temperature to pre-saturate the membrane filter and toreach a “pseudo-thermodynamic solubility”. Then the plate was put on a500 μL 96 multiwell plate cont

of CH₃CN in each well and centrifuged at 2000 rpm for 5 min. The soobtained solutions were then analyzed simultaneously by HPLC-UV withstandard solutions for quantification.

Example (A) Preparation of the MCM2 Conjugate

1.5 mg (0.045 μmol) of MCM2 protein (corresponding to residues 10-294 ofthe full length sequence, see Ishimi et al., 2001 Journal BiologicalChemistry, vol. 276, pages 42744-42752) were dissolved in 0.5 mL ofphosphate buffered saline solution (pH 7.2), pH value was adjusted to8.5 by addition of 55 μL of 1M NaHCO₃ (pH 8.5) and 0.5 mg of a compoundof formula (I), Comp. 8, were added from a 10 mg/mL DMSO solution. Thereaction was incubated for 1 h at room temperature then the reactionmixture was desalted on a NAP-10 column conditioned in phosphatebuffered saline solution and the fractions containing the protein werecollected and pooled.

Reacted MCM2 was characterized by HPLC/ESI mass spectrometry.

A reversed phase HPLC method (Poroshell C3 column 75×2.1 mm, 1100Agilent HPLC instrument) was coupled with an Agilent 1946 singlequadrupole mass spectrometry detector with an orthogonal ESI source.

The unreacted MCM2 protein showed a molecular weight of 33055 Da (FIG.1).

The reacted MCM2 protein showed a molecular weight of 34196 Da (FIG. 2).

The increase of 1139 Da in the molecular weight of the protein isindicative of the addition of a single molecule of Comp. 8 to the singlecysteine residue available on the MCM2 protein.

Example (B) Preparation of the Cysteine Conjugate

2 nmol of cysteine (Cys, MW 121 Da) have been reacted with 2 nmol of afunctionalized compound of formula (I), Comp. 17 (MW 1029 Da).

The reaction was incubated for 1 h at 21° C. in presence of boratebuffer 50 mM pH 8, DTPA (diethylene triamine pentaacetic acid) 2 mM,NaCl 50 mM, obtaining conjugate A1 (m/z=1150), and then it was analyzedby HPLC/ESI-MS using a reversed phase HPLC method (PLRP-S column 1000A 8μM 150×2.1 mm) on a 1100 Agilent HPLC instrument coupled with an Agilent1946 single quadrupole mass spectrometry detector with an orthogonal ESIsource (FIGS. 3 and 3 a).

Release of a Drug Moiety from a Conjugate

Example (C)

As an example, that is not intended to limit the scope of the invention,the release of a compound of formula (I) from the conjugate wasperformed in presence of cathepsin as reported below.

The conjugate A1 was incubated with 0.2 units of cathepsin B in sodiumacetate buffer pH 5.5 and 1 m

at 40° C. Then pH was adjusted to 7.4 by addition of Tris-HCl buffer andmaintained at room temperature for 1 h. Partial disappearance of theconjugate A1 and release of the corresponding compound of formula (I),Comp. 11, as well as of 9-bromo-camptothecin, confirms the breaking,first of the Z peptidic linker from the conjugate A1 (FIGS. 4 and 4 a),then the elimination of the self-immolative group W (FIGS. 5, 5 a and 5b).

The compounds of the present invention can be administered either assingle agents or, alternatively, in combination with known anticancertreatments such as radiation therapy or chemotherapy regimen, and/or incombination with cytostatic or cytotoxic agents, antibiotic-type agents,alkylating agents, antimetabolite agents, hormonal agents, immunologicalagents, interferon-type agents, cyclooxygenase inhibitors (e.g. COX-2inhibitors), matrix metalloproteinase inhibitors, telomerase inhibitors,tyrosine kinase inhibitors, anti-growth factor receptor agents, anti-HERagents, anti-EGFR agents, anti-angiogenesis agents (e.g. angiogenesisinhibitors), farnesyl transferase inhibitors, ras-raf signaltransduction pathway inhibitors, cell cycle inhibitors, other cdksinhibitors, tubulin binding agents, topoisomerase I inhibitors,topoisomerase II inhibitors, and the like.

If formulated as a fixed dose, such combination products employ thecompounds of this invention within the dosage range described below andthe other pharmaceutically active agent within the approved dosagerange.

Compounds of formula (I) may be used sequentially with known anticanceragents when a combination formulation is inappropriate.

The compounds of formula (I) of the present invention, suitable foradministration to a mammal, e.g., to humans, can be administered by theusual routes and the dosage level depends upon the age, the weight, theconditions of the patient and the administration route.

For example, a suitable dosage adopted for oral administration of acompound of formula (I) may range from about 1 to about 300 mg per dose,from 1 to 5 times daily. The compounds of the invention can beadministered in a variety of dosage forms, e.g., orally, in the form oftablets, capsules, sugar or film coated tablets, liquid solutions orsuspensions; rectally in the form of suppositories; parenterally, e.g.,subcutaneously, intramuscularly, or through intravenous and/orintrathecal and/or intraspinal injection or infusion.

The present invention also includes pharmaceutical compositionscomprising a compound of formula (I) or a pharmaceutically acceptablesalt thereof in association with a pharmaceutically acceptableexcipient, which may be a carrier or a diluent.

The pharmaceutical compositions containing the compounds of theinvention are usually prepared following conventional methods and areadministered in a suitable pharmaceutical form. For example, the solidoral forms may contain, together with the active compound, diluents,e.g., lactose, dextrose, saccharose, cellulose, corn starch or potatostarch; lubricants, e.g., silica, talc, stearic acid, magnesium orcalcium stearate, and/or polyethylene glycols; binding agents, e.g.,starches, arabic gum, gelatin, methylcellulose, carboxymethylcelluloseor polyvinyl pyrrolidone; disintegrating agents, e.g., starch, alginicacid, alginates or sodium starch glycolate; effervescing mixtures;dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates,laurylsulphates; and, in general, non-toxic and pharmacologicallyinactive substances used in pharmaceutical formulations. Thesepharmaceutical preparations may be manufactured in known manner, forexample, by means of mixing, granulating, tabletting, sugar-coating, orfilm-coating processes.

The liquid dispersions for oral administration may be, e.g., syrups,emulsions and suspensions. As an

syrups may contain, as carrier, saccharose or saccharose with glycerineand/or mannitol and sorbitol.

The suspensions and the emulsions may contain, as examples of carriers,natural gum, agar, sodium alginate, pectin, methylcellulose,carboxymethylcellulose or polyvinyl alcohol. The suspension or solutionsfor intramuscular injections may contain, together with the activecompound, a pharmaceutically acceptable carrier, e.g. sterile water,olive oil, ethyl oleate, glycols, e.g. propylene glycol, and, ifdesired, a suitable amount of lidocaine hydrochloride. The solutions forintravenous injections or infusions may contain, as a carrier, sterilewater or preferably they may be in the form of sterile, aqueous,isotonic, saline solutions or they may contain propylene glycol as acarrier. The suppositories may contain, together with the activecompound, a pharmaceutically acceptable carrier, e.g. cocoa butter,polyethylene glycol, a polyoxyethylene sorbitan fatty acid estersurfactant or lecithin.

With the aim at better illustrating the present invention, withoutposing any limitation to it, the following examples are now given.

EXAMPLES

The synthetic preparation of some compounds of formula (I) of theinvention is described in the following examples. The compounds of thepresent invention, as prepared according to the following examples, werealso characterized by ¹H-NMR and/or by Exact Mass data ESI(⁺).

¹H-NMR spectra were recorded at 28° C. on a Varian INOVA 400spectrometer operating at 400.5 MHz, at 25° C. on a Varian INOVA 500operating at 499.8 MHz and at 28° C. on a Varian INOVA 600 operating at599.9 MHz. Proton chemical shifts were referenced with respect to theresidual solvent signals (DMSO-d₆: 2.50 ppm; CH₃CN-d₃: 1.96 ppm). Dataare reported as follows: chemical shift (δ), multiplicity (s=singlet,d=doublet, t=triplet, q=quartet, br.s.=broad singlet, dd=doublet ofdoublets, m=multiplet), coupling constants (J, Hz), and number ofprotons.

ESI(+)mass spectra were obtained on a Q-Tof Ultima (Waters, Manchester,UK) directly connected with a 1100 micro-HPLC system (Agilent, PaloAlto, US) as previously described (M. Colombo, F. Riccardi-Sirtori, V.Rizzo, Rapid Commun. Mass Spectrom. 2004, 18, 511-517).

In the examples below as well as throughout the application, thefollowing abbreviations have the following meanings. If not defined, theterms have their generally accepted meanings.

ABBREVIATIONS DCC N,N′-dicyclohexylcarbodiimide DBU1,8-diazabicyclo[5.4.0]undec-7-ene DCM dichloromethane DIPEAN,N-diisopropyethylamine DMAP N,N-dimethylaminopyridine DMFN,N-dimethylformamide DMSO dimethyl sulfoxide EDCIN-ethyl-N′,N′-diisopropyl carbodiimide hydrochloride EDC1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Et₂O diethyl ether EtOAcethyl acetate EtOH ethanol HCl hydrochloric acid HOBt1H-benzotriazol-1-ol LiHMDS lithium bis(trimethylsilyl)amide MeOHmethanol Na₂SO₄ sodium sulfate NaHCO₃ sodium hydrogen carbonate NaOHsodium hydroxide TEA triethylamine TFA trifluoroacetic acid THFtetrahydrofuran mg milligrams mmol, μmol millimoles, micromoles M, mM,μM molar, millimolar, micromolar mL, μL milliliters, microliters μmmicrometers (M)Hz (mega)herz ppm parts-per-million h hour(s) minminute(s)

Example 1 Step 1b(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-ylhydrochloride (Ia), Comp. 1 [L=(IIk); W, Z, RM=null]

To a solution of 9-bromo-camptothecin (100 mg, 0.234 mmol) in anhydrousDMSO (6 mL), commercially available 4-nitrophenyl[(tert-butoxycarbonyl)amino]acetate (138.5 mg, 0.468 mmol) and DMAP (57mg, 0.468 mmol) were added. The mixture was stirred at room temperaturefor 3 days and then additional 2 equivalents of 4-nitrophenyl[(tert-butoxycarbonyl)amino]acetate and DMAP were added. Afteradditional two days, the mixture was diluted with DCM (50 mL), extractedwith water (3×20 mL) and washed with brine. The organic layer was driedwith Na₂SO₄, filtered and concentrated under vacuum. The product waspurified by flash chromatography (DCM/EtOAc 7/3) to provide theprotected intermediate as yellow powder that was dissolved in dioxane (5mL) and treated with HCl 4 M in dioxane (1 mL). The reaction was stirredat room temperature for 2 h and then the solvent was removed undervacuum. The resulting residue was triturated with Et₂O to afford thetitle product (90 mg, 69%).

ESI MS: m/z 484-486 (MH⁺)

¹H NMR (599.9 MHz, DMSO-d₆) δ ppm 0.95 (t, J=7.3 Hz, 3H), 2.14-2.24 (m,2H), 4.06-4.14 (m, 1H), 4.30-4.38 (m, 1H), 5.34-5.42 (m, 2H), 5.54-5.59(m, 2H), 7.34 (s, 1H), 7.81 (dd, J=8.6, 7.5 Hz, 1H), 8.11 (dd, J=7.5,0.9 Hz, 1H), 8.19 (d, J=8.6 Hz, 1H), 8.37 (br. s., 3H), 8.93 (s, 1H).

Example 2 Step 1e(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-ylpiperazine-1-carboxylate(Ia), Comp. 3 [L=(IIj); W, Z, RM=null]

To a solution of 9-bromo-camptothecin (100 mg, 0.234 mmol) in anhydrousTHF (10 mL), carbonyldiimidazole (304 mg, 1.87 mmol) was added. Themixture was stirred at 55° C. for 5 h and then was diluted with DCM (50mL), washed with diluted HCl (2×30 mL) and with brine (2×20 mL). Theorganic layer was dried with Na₂SO₄, filtered and concentrated undervacuum. The product was purified by flash chromatography (EtOAc 100%) toprovide the intermediate(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl1H-imidazole-1-carboxylate (84 mg, 69%).

ESI MS: m/z 521-523 (MH⁺)

¹H NMR (400.5 MHz, DMSO-d₆) δ ppm 1.02 (t, J=7.7 Hz, 3H), 2.29-2.35 (m,2H), 5.35 (s, 2H), 5.52

H), 7.18 (d, J=1.7 Hz, 1H), 7.33 (s, 1H), 7.73 (dd, J=7.3, 8.2 Hz, 1H),7.75 (d, J=1.7 Hz, 1H), 8.05 (dd, J=7.3, 1.7 Hz, 1H), 8.16 (dd, J=8.2,1.7 Hz, 1H), 8.52 (s, 1H), 8.88 (s, 1H).

To a solution of intermediate(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl1H-imidazole-1-carboxylate (30 mg, 0.0575 mmol), piperazine (7.5 mg,0.0863 mmol) in anhydrous CH₃CN (1.5 mL) was added and then stirred atroom temperature for 1.5 h until starting material was no longerdetected (HPLC-MS analysis). The mixture was diluted with DCM (50 mL),washed with water (2×30 mL) and then with brine (2×20 mL). The organiclayer was dried with Na₂SO₄, filtered and concentrated under vacuum toyield the title compound.

ESI MS: m/z 539-541 (MH⁺)

Analogously, the following compound has been prepared:

(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-ylmethyl[2-(methylamino)ethyl]carbamate hydrochloride (Ia), Comp. 7[L=(IIm); W, Z, RM=null]

(yield=88%)

ESI MS: m/z 541-543 (MH⁺)

Example 3 Step 2aN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[4-({[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}carbonyl)piperazin-1-yl]carbonyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(I), Comp. 5 [L=(IIj); W=(IIIc); Z=Citrulline-Valine; RM=(Va)]

A solution of(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-ylpiperazine-1-carboxylate (Comp. 3) (24.5 mg, 0.0457 mmol) andN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-ornithinamide(MC-Val-Cit-PABC-PNP), prepared as reported in EP0624377A2 andEP2357006A2 (33 mg, 0.0457 mmol), in anhydrous DMSO (1.0 mL) was stirredat room temperature for 2 h until starting material was no longerdetected (HPLC-MS analysis). Et₂O was added to the mixture and twophases were formed. The upper phase was removed and this sequence wasrepeated until an amorphous solid was formed on the bottom of the flask.The solid was purified by flash chromatography (EtOAc/MeOH 90/10) toprovide the title compound (1.5 mg, 3%).

ESI MS: m/z 1137-1139 (MH⁺)

¹H NMR (499.8 MHz, DMSO-d₆) δ ppm 0.77-0.88 (m, 6H), 0.90 (2×t, J=7.6Hz, 3H), 1.12-1.28 (m, 2H), 1.30-1.40 (m, 2H), 1.89-1.52 (m, 4H),1.53-1.75 (m, 2H), 1.91-2.01 (m, 1H); 2.06-2.20 (m, 4H), 2.88-3-06 (m,2H), 3.12-3.74 (m partially overlapped by water signal, 10H), 4.15-4.21(m, 1H), 4.32-4.41 (m, 1H), 5.03 (br. s., 2H), 5.34 (s, 2H), 5.41 (br.s., 2H), 5.40-5.50 (m, 2H), 5.94-6.02 (br. s., 1H), 7.00 (s, 2H), 7.12(s, 1H), 7.25-7.36 ((br. s., 2H), 7.52-7.67 (br. s., 2H), 7.74-7.84 (m,2H), 8.05-8.13 (d, J=6.9 Hz, 1H), 8.10 (br. s., 1H), 8.14-8.25 (m, 1H),8.90 (s, 1H), 10.01 (br. s., 1H).

Analogously, starting from Comp. 7 the following compound has beenprepared:

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[{2-[({[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide(I), Comp. 8. [L=(IIm); W=(IIIc); Z=Citrullina-Valine; RM=(Va)]

ESI MS: m/z 1139-1141 (MH⁺)

¹H NMR (499.8 MHz, DMSO-d₆, the spectrum is a mixture of rotationalisomers) δ ppm 0.75-0.87 (m, 6H), 0.87-0.94 (m, 3H), 1.12-1.22 (m, 2H),1.29-1.40 (m, 2H), 1.40-1.63 (m, 4H), 1.53-1.73 (m, 2H), 1.87-2.02 (m,2H), 2.04-2.22 (m, 4H), 2.88-3.05 (m, 2H), 2.70, 2.72, 2.77, 2.80 (4×s,rotational isomers, 3H), 2.98, 3.02, 3.09, 3.12 (4×s, rotationalisomers, 3H), 3.24-3.63 (m, partially overlapped by water signal, 6H),4.13-4.22 (m, 1H), 4.28-4.42 (m, 1H), 4.83, 5.00, 5.09 (3×s, rotationalisomers, 2H), 5.28-5.50 (m, 4H), 5.41 (br. s., 2H), 6.00 (br. s., 1H),7.00 (s, 1H), 7.06-7.11 (m, 1H), 7.07, 7.21, 7.23, 7.31, 7.40, 7.42,7.47, 7.54 (8×d, J=8.6 Hz, rotational isomers, 4H), 7.74-7.84 (m, 2H),8.03-8.23 (m, 3H), 8.84, 8.88, 8.89 (3×s, rotational isomers, 1H), 9.91,9.94, 9.996, 9.97 (4×br. s., rotational isomers, 1H).

Example 4 Step 8b(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-ylL-phenylalanyl-L-leucylglycinate (I), Comp. 2 [L=null; W=null;Z=Glycine-Leucine-Phenylalanine; RM=null]

To a solution of 9-bromo-camptothecin (100 mg, 0.234 mmol) in anhydrousDMSO (10 mL), 4-nitroph

phenylalanyl-L-leucylglycinate (326.6 mg, 0.468 mmol) and DMAP (57 mg,0.468 mmol) were added. The mixture was stirred at room temperature for1 day and then two additional equivalent of 4-nitrophenylN-trityl-L-phenylalanyl-L-leucylglycinate and DMAP were added. Afteradditional two days, the mixture was diluted with DCM, (20 mL) extractedwith water (3×10 mL) and washed with brine. The organic layer was driedwith Na₂SO₄, filtered and concentrated under vacuum. The product waspurified by flash chromatography (DCM/EtOAc 7/3) to provide a brownsolid protected intermediate. The intermediate was dissolved in amixture of acetic acid-water (75-25, 10 mL) and the reaction was stirredat room temperature for 1 h. The solvent was removed under vacuum andthe crude product was dissolved in DCM (20 mL), washed with a saturatedsolution of NaHCO₃ (2×20 mL), water and brine. The resulting residue waspurified by flash chromatography (DCM/MeOH from 99/1 to 97/3) to providethe title compound (110 mg, 63%). ESI MS: m/z 744-746 (MH⁺)

¹H NMR (499.8 MHz, DMSO-d₆) δ ppm 0.81 (d, J=6.1 Hz, 6H), 0.92 (t, J=7.4Hz, 3H), 1.42-1.48 (m, 2H), 1.49-1.55 (m, 1H), 1.68 (br. s., 2H),2.08-2.18 (m, 2H), 2.57 (dd, J=13.3, 8.2 Hz, 1H), 2.87 (dd, J=13.3, 4.5Hz, 1H), 3.38 (dd, J=8.2, 4.5 Hz, 1H), 3.98-4.20 (m, 2H), 4.33-4.41 (m,1H), 5.32 (s, 2H), 5.51 (s, 2H), 7.11-7.24 (m, 6H), 7.77 (dd, J=8.4, 7.6Hz, 1H), 7.96 (d, J=8.7 Hz, 1H), 8.09 (d, J=7.4 Hz, 1H), 8.19 (d, J=8.6Hz, 1H), 8.50 (t, J=5.9 Hz, 1H), 8.88 (s, 1H).

Example 5(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-ylN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-L-leucylglycinate(I), Comp. 4 [L=null; W=null; Z=Glycine-Leucine-Phenylalanine; RM=(Va)]

A solution of((4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-ylL-phenylalanyl-L-leucylglycinate (Comp. 2) (50 mg, 0.0671 mmol) andN-ε-Maleimidocaproyl oxysuccinimide ester (prepared as reported inHelvetica chimica Acta, 1975, 58, 2, 531-541) (31 mg, 0.101 mmol) in amixture of DCM/CH₃CN (1/1 2 mL) was stirred at room temperature for 15 huntil starting material was no longer detected (HPLC-MS analysis). Themixture was diluted with DCM (50 mL), washed with water (2×20 mL) andthen with brine (2×20 mL). The organic layer was dried with Na₂SO4,filtered and concentrated under vacuum. The product was purified byflash chromatography (DCM/MeOH 97/3) to provide the title compound (34mg 55%). ESI MS: m/z 937-939 (MH⁺)

¹H NMR (499.8 MHz, CH₃CN-d₃)) δ ppm 0.81 (d, J=5.9 Hz, 3H), 0.83 (d,J=5.9 Hz, 3H), 0.98 (t, J=1.07-1.15 (m, 1H), 1.38-1.48 (m, 4H),1.50-1.59 (m, 3H), 2.02 (t, J=7.6 Hz, 2H), 2.09-2.25 (m, 2H), 2.71 (dd,J=14.0, 8.2 Hz, 1H), 2.87 (dd, J=14.0, 5.6 Hz, 1H), 3.36 (t, J=7.2 Hz,2H), 3.97 (dd, J=17.9, 5.9 Hz, 1H), 4.13 (dd, J=17.9, 6.3 Hz, 1H),4.29-4.35 (m, 1H), 4.39-4.45 (m, 1H), 5.09-5.21 (m, 2H), 5.37 (d, J=17.0Hz, 1H), 5.57 (d, J=17.0 Hz, 1H), 6.50 (d, J=7.4 Hz, 1H), 6.71 (s, 2H),6.80 (d, J=8.2 Hz, 1H), 6.99 (t, J=6.0 Hz, 1H), 7.02-7.06 (m, 2H),7.14-7.20 (m, 3H), 7.21 (s, 1H), 7.71 (dd, J=8.5, 7.4 Hz, 1H), 7.99 (d,J=7.4 Hz, 1H), 8.16 (d, J=8.5 Hz, 1H), 8.80 (s, 1H).

Analogously, starting from compound 3 the following compound has beenprepared:

(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]piperazine-1-carboxylate(I), Comp. 6 [L=(IIj); W=null; Z=null; RM=(Va)]

ESI MS: m/z 732-734 (MH⁺)

¹H NMR (499.8 MHz, CH₃CN-d₃)) δ ppm 0.97 (t, J=7.6 Hz, 3H), 1.20-1.34(m, 2H), 1.47-1.60 (m, 4H), 2.10-2.22 (m, 2H), 2.22-2.38 (m, 2H),3.16-3.74 (m, 10H), 5.21-5.30 (m, 2H), 5.37 (d, J=17 Hz, 1H), 5.52 (d,J=17 Hz, 1H), 6.68-6.76 (m, 2H), 7.14 (s, 1H), 7.72 (dd, J=8.5, 7.4 Hz,1H), 7.98 (d, J=7.4 Hz, 1H), 8.14 (d, J=8.5 Hz, 1H), 8.85 (s, 1H).

Example 6 Step 5, Deprotection, Step 6b, Deprotection, Step 7 Step 5(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-[(tert-butoxycarbonyl)amino]-3,3-dimethylbutanoate

To a solution of 9-bromo-camptothecin (28 mg, 0.066 mmol) in anhydrousDMSO (0.5 mL), 4-nitrophenyl4-[(tert-butoxycarbonyl)amino]-3,3-dimethylbutanoate (35 mg, 0.1 mmol)and DMAP (16 mg, 0.13 mmol) were added. The mixture was stirred at roomtemperature for 1 day and then 1.5 additional equivalents of4-nitrophenyl 4-[(tert-butoxycarbonyl)amino]-3,3-dimethylbutanoate andDMAP were added. Additional 2 equivalents of DMAP were added after 3days and 6 days. The mixture was diluted with DCM, (20 mL), extractedwith water (3×10 mL) and washed with brine. The organic layer was driedwith Na₂SO₄, filtered and concentrated under vacuum. The

purified by flash chromatography (DCM/acetone 95/5) to provide the titlecompound (10 mg, 24%).

ESI MS: m/z 640-642 (MH⁺)

¹H NMR (499.8 MHz, DMSO-d₆) δ ppm 0.91 (s, 3H) 0.92 (t, J=7.4 Hz, 3H)0.97 (s, 3H) 1.33-1.41 (m, 9H) 2.11-2.19 (m, 2H) 2.26 (d, J=13.8 Hz, 1H)2.47 (d, J=13.8 Hz, 1H) 2.87-2.93 (m, 2H) 5.30-5.40 (m, 2H) 5.45-5.54(m, 2H) 6.89 (t, J=6.2 Hz, 1H) 7.12 (s, 1H) 7.77-7.83 (m, 1H) 8.09 (d,J=7.2 Hz, 1H) 8.20 (d, J=8.4 Hz, 1H) 8.90 (s, 1H).

Analogously, starting from 4-nitrophenyl5-[(tert-butoxycarbonyl)amino]-4,4-difluoropentanoate (prepared asreported in WO03/014069) the following compound has been prepared:

(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl5-[(tert-butoxycarbonyl)amino]-4,4-difluoropentanoate

ESI MS: m/z 662-664 (MH⁺)

¹H NMR (499.7 MHz, CD₃CN) δ ppm 0.97 (t, J=7.4 Hz, 3H) 1.32 (m, 9H)2.07-2.32 (m, 4H) 2.65-2.83 (m, 2H) 3.43-3.52 (m, 2H) 5.21-5.29 (m, 2H)5.39 (d, J=17.0 Hz, 1H) 5.56 (d, J=17.0 Hz, 1H) 5.83 (br. s., 1H) 7.14(s, 1H) 7.70-7.76 (m, 1H) 8.00 (d, J=7.4 Hz, 1H) 8.16 (d, J=8.5 Hz, 1H)8.86 (s, 1H).

(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl(1-{[(tert-butoxycarbonyl)amino]methyl}cyclohexyl)acetate

A suspension of 9-bromo-camptothecin (10 mg, 0.023 mmol), scandiumtriflate (6.8 mg, 0.138 mmol) and DMAP (8.5 mg, 0.07 mmol) in anhydrousDCM (8 mL) was cooled to 0° C., followed by the addition of4-nitrophenyl (1-{[(tert-butoxycarbonyl)amino]methyl}cyclohexyl)acetate(27 mg, 0.070 mmol). The reaction mixture was stirred at 0° C. for 30min and at room temperature for 36 h, then it was filtered and thefiltrate was washed with 10 mL of 0.1 N hydrochloric acid, 10 mL of 0.1M NaHCO₃, 20 mL of distilled water and then dried over anhydrous Na₂SO₄.The organic solvent was removed under reduced pressure and the residuepurified by flash chromatography on silica gel (eluent: DCM:acetone(95:5 v/v) to give the title compound (9 mg) (TLC on kieselgel plate,eluent:|

(95:5 v/v). Rf=0.25)

ESI MS: m/z 680-682 (MH⁺)

¹H NMR (499.7 MHz, DMSO-d₆) δ ppm 0.92 (t, J=7.4 Hz, 3H) 1.20-1.51 (m,10H) 1.37 (s, 9H) 2.07-2.21 (m, 2H) 2.36 (d, J=13.8 Hz, 1H) 2.56 (d,J=13.8 Hz, 1H) 3.09 (d, J=6.0 Hz, 2H) 5.31-5.40 (m, 2H) 5.46-5.53 (m,2H) 6.70 (t, J=6.0 Hz, 1H) 7.18 (s, 1H) 7.79-7.82 (m, 1H) 8.09 (d, J=7.4Hz, 1H) 8.18 (d, J=8.5 Hz, 1H) 8.90 (s, 1H).

Deprotection(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-amino-3,3-dimethylbutanoate hydrochloride (Ic), Comp. 9 [L=null;W=(IIIa); Z=null; RM=null]

(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-[(tert-butoxycarbonyl)amino]-3,3-dimethylbutanoate (10 mg, 0.016 mmol)was dissolved in 4N HCl in dioxane (0.5 mL) and the reaction was stirredat room temperature for 30 min. The solvents was removed under vacuumand the resulting residue was purified by flash chromatography (DCM/MeOHfrom 97/3 to 93/7) to provide the title compound (1.9 mg, 19%).

ESI MS: m/z 540-542 (MH⁺)

¹H NMR (499.8 MHz, DMSO-d₆) δ ppm 0.93 (t, J=7.4 Hz, 3H) 1.00 (s, 3H)1.02 (s, 3H) 2.12-2.20 (m, 2H) 2.43 (d, J=14.0 Hz, 1H) 2.60 (d, J=14.0Hz, 1H) 2.58-2.71 (m, 2H) 3.32 (m overlapped by water signal, 2H)5.30-5.40 (m, 2H) 5.47-5.55 (m, 2H) 7.12 (s, 1H) 7.76-7.84 (m, 1H) 8.10(d, J=7.7 Hz, 1H) 8.20 (d, J=8.4 Hz, 1H) 8.23 (br. s., 3H) 8.91 (s, 1H).

Analogously, the following compounds have been prepared:

(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl5-amino-4,4-difluoropentanoate hydrochloride (Ic), Comp. 10 [L=null;W=(IIIa); Z=null; RM=null]

ESI MS: m/z 562-564 (MH⁺)

¹H NMR (499.7 MHz, DMSO-d₆) δ ppm 0.93 (t, J=7.4 Hz, 3H) 2.13-2.21 (m,2H) 2.26-2.38 (m, 2

(m, 2H) 3.41-3.52 (m, 2H) 5.32-5.41 (m, 2H) 5.48-5.55 (m, 2H) 7.18 (s,1H) 7.78-7.84 (m, 1H) 8.10 (d, J=7.4 Hz, 1H) 8.22 (d, J=8.4 Hz, 1H) 8.38(br. s., 3H) 8.91 (s, 1H).

4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl[1-(aminomethyl)cyclohexyl]acetatehydrochloride (Ic), Comp. 11 [L=null; W=(IIIa); Z=null; RM=null]

ESI MS: m/z 580-582 (MH⁺)

¹H NMR (500 MHz, DMSO-d₆) δ ppm 0.94 (t, J=7.4 Hz, 3H) 1.25-1.66 (m,10H) 2.09-2.20 (m, 2H) 2.60 (d, J. 14.0 Hz, 1H) 2.77-2.97 (m, 3H)5.32-5.41 (m, 2H) 5.47-5.55 (m, 2H) 7.16 (s, 1H) 7.79-7.83 (m, 1H) 8.10(d, J=7.4 Hz, 1H) 8.14 (br.s., 3H) 8.18 (d, J=8.5 Hz, 1H) 8.92 (s, 1H).

Step 6bN-(tert-butoxycarbonyl)-L-valyl-N-[4-({[(5-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-difluoro-5-oxopentyl)carbamoyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide

N-(tert-butoxycarbonyl)-L-valyl-N⁵-carbamoyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-ornithinamide(prepared as reported in WO2005/112919) (8.3 mg, 0.0128 mmol) and(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl5-amino-4,4-difluoropentanoate hydrochloride (7 mg, 0.0117 mmol) weredissolved in DMF (0.15 mL) and anhydrous DCM (1 mL) and DMAP (3.3 mg,0.0269 mmol) was added. The reaction mixture was stirred at roomtemperature for one day in argon atmosphere, then poured into water,aqueous acetic acid and DCM. The organic phase was separated and washedwith water. The organic solvent was removed under reduced pressure andthe residue was purified by flash chromatography on silica gel (eluent:DCM:MeOH (95:5 v/v) to give the title compound (TLC on kieselgel plate,eluting system DCM:MeOH 0.25)

ESI MS: m/z 1067-1069 (MH⁺)

Analogously, the following compounds can be prepared:

N-(tert-butoxycarbonyl)-L-valyl-N-[4-({[(4-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-dimethyl-4-oxobutyl)carbamoyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide

ESI MS: m/z 1045-1047 (MH⁺)

N-(tert-butoxycarbonyl)-L-valyl-N-(4-{[({[1-(2-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2-oxoethyl)cyclohexyl]methyl}carbamoyl)oxy]methyl}phenyl)-N⁵-carbamoyl-L-ornithinamide

ESI MS: m/z 1085-1087 (MH⁺)

N-(tert-butoxycarbonyI)-L-valyl-N-{[1-(2-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2-oxoethyl)cyclohexyl]methyl}-N⁵-carbamoyl-L-ornithinamide

N-(tert-butoxycarbonyl)-L-valyl-N⁵-carbamoyl-L-ornithine (prepared asreported in WO2005/112919) (

mmol), DCC (22 mg, 0.106 mmol) and HOBt (17 mg, 0.106 mmol) weredissolved in dry THF (2 mL) and stirred at room temperature for 3 h. Tothe solution was then added4(S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl[1-(aminomethyl)cyclohexyl]acetatehydrochloride (Comp 11) (43 mg, 0.073 mmol) and 4-dimethylaminopyridine(23 mg, 0.188 mmol). The reaction was stirred at room temperature for 40min then, 20 mL of water were added. The water solution was extractedwith DCM (20 mL) and then washed twice with water (10 mL). The organicphase was separated, dried over Na₂SO₄, the solvent was removed underreduced pressure and the crude reside was purified by flashchromatography on silica gel (eluant DCM:MeOH, 98:2 to 95:5 v/v) toyield the title compound as a white powder (40 mg, yield 58%).

ESI MS: m/z 936-938 (MH⁺)

¹H NMR (499.8 MHz, CH₃CN-d₃) δ ppm 0.80 (d, J=6.8 Hz, 3H), 0.85 (d,J=6.8 Hz, 3H), 0.97 (t, J=7.4 Hz, 3H), 1.29-1.58 (m, 13H), 1.37 (s, 9H),1.68-1.74 (m, 1H), 1.94-2.02 (m, 1H), 2.15-2.24 (m, 2H), 2.44 (d, J=13.9Hz, 1H), 2.56 (d, J=13.9 Hz, 1H), 2.90-2.98 (m, 1H), 3.17-3.29 (m, 2H),3.36 (dd, J=13.9, 7.0 Hz, 1H), 3.79-3.86 (m, 1H), 4.44 (m, 1H), 4.68(br.s., 2H), 5.17 (br. s., 1H), 5.21-5.31 (m, 2H), 5.39 (d, J=16.9 Hz,1H), 5.50 (d, J=7.5 Hz, 1H), 5.57 (d, J=16.9 Hz, 1H), 6.90 (br. s., 1H),6.96 (d, J=7.9 Hz, 1H), 7.21 (s, 1H), 7.72 (dd, J=8.5, 7.5 Hz, 1H), 8.00(d, J=7.5 Hz, 1H), 8.15 (d, J=8.5 Hz, 1H), 8.86 (s, 1H).

Analogously the following compounds can be prepared:

N-(tert-butoxycarbonyl)-L-valyl-N-(4-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-dimethyl-4-oxobutyl)-N⁵-carbamoyl-L-ornithinamide

ESI MS: m/z 896-898 (MH⁺)

N-(tert-butoxycarbonyl)-L-valyl-N-(5-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-difluoro-5-oxopentyl)-N⁵-carbamoyl-L-ornithinamide

ESI MS: m/z 918-920 (MH⁺)

Deprotection

Deprotection of the intermediates prepared in step 6a was carried outunder similar conditions reported above giving the compounds below:

L-valyl-N-[4-({[(5-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-difluoro-5-oxopentyl)carbamoyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide(Id), Comp. 12 [L=null; W=(IIIj); Z=Citrulline-Valine; RM=null]

ESI MS: m/z 967-969 (MH⁺)

L-valyl-N-[4-({[(4-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-dimethyl-4-oxobutyl)carbamoyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide(Id), Comp. 13 [L=null; W=(IIIj); Z=Citrulline-Valine; RM=null]

ESI MS: m/z 945-947 (MH⁺)

L-valyl-N-(4-{[({[1-(2-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2-oxoethyl)cyclohexyl]methyl}carbamoyl)oxy]methyl}phenyl)-N⁵-carbamoyl-L-ornithinamide(Id), Comp. 14 [L=null; W=(IIIj); Z=Citrulline-Valine; RM=null]

ESI MS: m/z 985-9847 (MH⁺)

L-valyl-N-{[1-(2-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2-oxoethyl)cyclohexyl]methyl}-N⁵-carbamoyl-L-ornithinamide(Id), Comp. 25 [L=null; W=(IIIa); Z=Citrulline-Valine; RM=null]

ESI MS: m/z 936-838 (MH⁺)

L-valyl-N-(4-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-dimethyl-4-oxobutyl)-N⁵-carbamoyl-L-ornithinamide(Id), Comp. 26 [L=null; W=(IIIa); Z=Citrulline-Valine; RM=null]

ESI MS: m/z 796-799 (MH⁺)

L-valyl-N-(5-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-difluoro-5-oxopentyl)-N⁵-carbamoyl-L-ornithinamide(Id), Comp. 27 [L=null; W=(IIIa); Z=Citrulline-Valine; RM=null]

ESI MS: m/z 818-820 (MH⁺)

Step 7N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{[1-(2-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2-oxoethyl)cyclohexyl]methyl}-N⁵-carbamoyl-L-ornithinamide(I), Comp. 17 [L=null; W=(IIIa); Z=Citrulline-Valine; RM=(Va)]

6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (12 mg, 0.053mmol), DCC (13.3 mg, 0.063 mmol) and HOBt (10 mg, 0.063 mmol) weredissolved in dry THF (2 mL) and stirred at room temperature for 3 h. Thesuspension was filtered and to the THF solutionL-valyl-N-{[1-(2-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-

pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2-oxoethyl)cyclohexyl]methyl}-N⁵-carbamoyl-L-ornithinamide(Comp. 25) (15 mg, 0.018 mmol) and 4-dimethylaminopyridine (6.6 mg,0.055 mmol) were added dropwise. The reaction was stirred at roomtemperature for 20 min then, 20 mL of water were added. The watersolution was extracted with DCM (20 mL) and then washed twice with water(10 mL). The organic phase was separated, dried over Na₂SO₄, the solventwas removed under reduced pressure and the crude reside was purified byflash chromatography on silica gel (eluant DCM:MeOH, 97:3 to 95:5 v/v)to yield the title compound as a white powder (14 mg, yield 78%).

ESI MS: m/z 1029-1031 (MH⁺)

¹H NMR (499.8 MHz, CH₃CN-d₃) ppm 0.83 (d, J=6.4 Hz, 3H), 0.84 (d, J=6.4Hz, 3H), 0.97 (t, J=7.4 Hz, 3H), 1.19-1.59 (m, 19H), 1.69-1.77 (m, 1H),1.99-2.06 (m, 1H), 2.13-2.24 (m, 4H), 2.43 (d, J=13.8 Hz, 1H), 2.57 (d,J=13.8 Hz, 1H), 2.94-3.01 (m, 1H), 3.11-3.20 (m, 1H), 3.24 (dd, J=13.6,5.9 Hz, 1H), 3.31 (dd, J=13.6, 6.7 Hz, 1H), 3.39 (t, J=7.1 Hz, 2H), 4.08(dd, J=8.0, 5.9 Hz, 1H), 4.32-4.41 (m, 1H), 4.68 (br.s., 2H), 5.19 (br.s., 1H), 5.22-5.32 (m, 2H), 5.39 (d, J=17.0 Hz, 1H), 5.57 (d, J=17.0 Hz,1H), 6.58 (d, J=8.0 Hz, 1H), 6.70 (s, 2H), 6.86 (m, 1H), 7.02 (d, J=7.9Hz, 1H), 7.22 (s, 1H), 7.73 (dd, J=8.5, 7.5 Hz, 1H), 8.00 (d, J=7.5 Hz,1H), 8.15 (d, J=8.5 Hz, 1H), 8.87 (s, 1H).

Analogously, the following compounds can be prepared:

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-(5-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-difluoro-5-oxopentyl)-N⁵-carbamoyl-L-ornithinamide(I), Comp. 16 [L=null; W=(IIIa); Z=Citrulline-Valine; RM=(Va)]

ESI MS: m/z 1011-1013 (MH⁺)

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-(4-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-dimethyl-4-oxobutyl)-N⁵-carbamoyl-L-ornithinamide(I), Comp. 18 [L=null; W=(IIIa); Z=Citrulline-Valine; RM=(Va)]

ESI MS: m/z 989-991 (MH⁺)

N-[6-(2,5-dioxopyrrolidin-1-yl)hexanoyl]-valyl-N-[4-({[(5-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-difluoro-5-oxopentyl)carbamoyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide(I), Comp. 15 [L=null; W=(IIIj); Z=Citrulline-Valine; RM=(Va)]

ESI MS: m/z 1162-1164 (MH⁺)

N-(3-carboxypropanoyl)-L-valyl-N-(5-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-difluoro-5-oxopentyl)-N⁵-carbamoyl-L-ornithinamide(I), Comp. 19 [L=null; W=(IIIa); Z=Citrulline-Valine; RM=(Vn)]

ESI MS: m/z 918-920 (MH⁺)

N-(3-carboxypropanoyl)-L-valyl-N-{[1-(2-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2-oxoethyl)cyclohexyl]methyl}-N⁵-carbamoyl-L-ornithinamide(I), Comp. 20 [L=null; W=(IIIa); Z=Citrulline-Valine; RM=(Vn)]

ESI MS: m/z 936-938 (MH⁺)

N-(3-carboxypropanoyl)-L-valyl-N-(4-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-dimethyl-4-oxobutyl)-N⁵-carbamoyl-L-ornithinamide(I), Comp. 21 [L=null; W=(IIIa); Z=Citrulline-Valine; RM=(Va)]

ESI MS: m/z 896-898 (MH⁺)

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-N-(5-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-difluoro-5-oxopentyl)-L-leucinamide(I), Comp. 22 [L=null; W=(IIIa); Z=Glicine-Leucine-Phenylalanine;RM=(Va)]

ESI MS: m/z 1015-1017 (MH⁺)

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-N-{[1-(2-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2-oxoethyl)cyclohexyl]methyl}-L-leucinamide(I), Comp. 23 [L=null; W=(IIIa); Z=Glicine-Leucine-Phenylalanine;RM=(Va)]

ESI MS: m/z 1033-12035 (MH⁺)

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-N-(4-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-dimethyl-4-oxobutyl)-L-leucinamide(I), Comp. 24 [L=null; W=(IIIa); Z=Glicine-Leucine-Phenylalanine;RM=(Va)]

ESI MS: m/z 993-995 (MH⁺)

Synthesis of Intermediates Methyl 3,3-dimethyl-4-nitrobutanoate

Ethyl 3,3-dimethylacrylate (6.1 mL, 46.7 mmol) was dissolved innitromethane (13.5 mL) and DBU (7.6 mL, 50.8 mmol) was added. Themixture was stirred at room temperature for 24 h. Et₂O (40 mL) was addedand the solution was washed with 1M HCl (2×20 mL) and then with water(20 mL). The organic phase was dried over Na₂SO₄ and the solvent wasevaporated under reduced pressure. The product was purified by flashchromatography (DCM) to provide the title compound (6.5 g, 79% yield) ascolorless oil.

¹H NMR (499.8 MHz, DMSO-d₆) δ ppm 1.08 (s, 6H) 2.44 (s, 2H) 3.60 (s, 3H)4.58 (s, 2H)

Analogously, starting from ethyl cyclohexylideneacetate the followingcompound has been prepared:

ethyl[1-(nitromethyl)cyclohexyl]acetate

4-amino-3,3-dimethylbutanoic acid hydrochloride

3,3-Dimethyl-4-nitro-butyric acid methyl ester (1.0 g, 5.7 mmol) wasdissolved in THF (40 mL) followed by addition of 10% palladium onactivated charcoal (400 mg) and a solution of ammonium formate (10 mL,25% w/w in water). The reaction mixture was stirred, under nitrogenatmosphere for 48 h. Palladium was removed by filtration and thefiltrate was extracted with DCM (2×40 mL). The organic phases were driedover Na₂SO₄ and the solvent was evaporated under reduced pressure togive 4,4-dimethyl-2-pyrrolidinone intermediate. This compound was addedto a mixture of concentrated HCl (15 mL) and water (15 mL) and theresulting mixture refluxed at 110° C. for 20 h. After cooling to roomtemperature the mixture was evaporated to give4-amino-3,3-dimethyl-butyric acid hydrochloride (560 mg, 52% yield) as abrown solid.

¹H NMR (499.8 MHz, DMSO-d₆) δ ppm 0.99 (s, 6H) 2.2.26 (s, 2H) 2.78 (q,2H)

Analogously, starting from ethyl[1-(nitromethyl)cyclohexyl]acetate thefollowing compound has been prepared:

[1-(aminomethyl)cyclohexyl]acetic acid hydrochloride

¹H NMR (499.7 MHz, DMSO-d₆) δ ppm 1.25-1.50 (m, 10H) 2.40 (s, 2H) 2.90(s, 2H) 7.99 (br. s., 3H) 12.36 (br. s., 1H).

4-[(tert-butoxycarbonyl)amino]-3,3-dimethylbutanoic acid

Di-tert-butyl dicarbonate (600 mg, 2.85 mmol) was added to a mixture of4-amino-3,3-dimethyl-butyric acid hydrochloride (400 mg, 2.38 mmol) intert-butyl alcohol (2.4 mL) and 2N NaOH (2.4 mL). The reaction wasstirred at room temperature for 4 h and acetic acid (0.18 mL) was added.The mixture was extracted with EtOAc (3×10 mL), the combined organicphases were dried over Na₂SO₄ and then evaporated under reducedpressure. The residue was purified by flash chromatography (DCM/MeOH98.5/1.5) to provide the title compound (110 mg, 20%).

¹H NMR (499.8 MHz, DMSO-d₆) δ ppm 0.89 (s, 6H) 1.38 (s, 9H) 2.06 (s, 2H)2.86 (d, 2H)

Analogously, starting from [1-(aminomethyl)cyclohexyl]acetic acidhydrochloride the following compound has been prepared:

(1-{[(tert-butoxycarbonyl)amino]methyl}cyclohexyl)acetic acid

¹H NMR (400.5 MHz, DMSO-d₆) δ ppm 1.21-1.53 (m, 10H) 1.37 (s, 9H) 2.15(s, 2H) 3.03 (d, J=6.5 Hz, 2H) 6.55 (t, J=6.5 Hz, 1H) 11.96 (br. s.,1H).

4-nitrophenyl 4-[(tert-butoxycarbonyl)amino]-3,3-dimethylbutanoate

To a solution of 4-[(tert-butoxycarbonyl)amino]-3,3-dimethylbutanoicacid (90 mg, 0.389 mmol) and 4-nitrophenol (54 mg, 0.389 mmol) inanhydrous THF (1 mL), DCC (96.3 mg, 0.467 mmol) was added. The reactionwas stirred for 2 h at room temperature and then the white solid formedwas removed by filtration. The filtrate was evaporated under reducedpressure and finally purified by flash chromatography (DCM) to providethe title compound (80 mg, 58%).

ESI MS: m/z 353 (MH⁺)

¹H NMR (499.8 MHz, DMSO-d₆) δ ppm 0.99 (s, 6H) 1.39 (s, 9H) 2.49 (signalpartially overlapped by DMSO signal, 2H) 2.95 (d, J=6.5 Hz, 2H) 6.99 (d,J=6.5 Hz, 1H) 7.39-7.49 (m, 2H) 8.27-8.34 (m, 2H).

Analogously, starting from(1-{[(tert-butoxycarbonyl)amino]methyl}cyclohexyl)acetic acid thefollowing compound has been prepared:

4-nitrophenyl (1-{[(tert-butoxycarbonyl)amino]methyl}cyclohexyl)acetate

ESI MS: m/z 393 (MH⁺)

¹H NMR (499.7 MHz, DMSO-d₆) δ ppm 1.09-1.57 (m, 10H) 1.37 (s, 9H) 2.55(s, 2H) 3.10 (d, J=6.4 Hz, 2H) 6.86 (t, J=6.4 Hz, 1H) 7.44 (d, J=9.0 Hz,2H) 8.30 (m, d, J=9.0 Hz, 2H).

1. A compound of formula (I)

wherein: L is null or a conditionally-cleavable moiety; W is null or aself-immolative system, comprising one or more self-immolative groups; Zis null or a peptidic, non peptidic or hybrid-peptidic and nonpeptidic-linker; RM is null or a reactive moiety that can be attached toone or more of L, W or Z groups, or RM is attached to oxygen when L, Wand Z are all null; provided that at least one of L, W, Z and RM is notnull; or a pharmaceutically acceptable salt thereof.
 2. A compound offormula (I) according to claim 1, wherein: the conditionally-cleavablemoiety L is independently null or a group selected from:

wherein: R1, R2 and R3 are, each independently, null, hydrogen, hydroxyor an optionally substituted group selected from linear or branchedC₁-C₄ alkyl, linear or branched C₁-C₄ hydroxyalkyl, linear or branchedC₁-C₄ sulfhydrylalkyl, linear or branched C₁-C₄ aminoalkyl, linear orbranched C₁-C₄ alkylcarbonyl and linear or branched C₁-C₄alkoxycarbonyl; each of n is independently an integer from 0 to 2; andn1 is an integer from 0 to 5; the self-immolative system W isindependently null or a group selected from:

wherein: one of R1 and R2 is null and the other is as defined above; R3is as defined above; R4 and R5 are, each independently, halogen, methyl,ethyl or linear or branched C₁-C₄ hydroxyalkyl; m is an integer from 0to 3; A is C₁-C₃ alkyl, CH₂NH, NH or N—R4, wherein R4 is as definedabove; and R12 and R13 are, each independently, hydrogen, halogen,methyl, ethyl, linear or branched C₁-C₄ hydroxyalkyl, linear or branchedC₁-C₄ haloalkyl, or R12 and R13, taken together, form a 3- to 6-memberedcarbocycle; the Z linker is independently null or Z1, wherein Z1 is adipeptide or a tripeptide, linked via its C-terminus to W, or to L whenW is null, or to oxygen when W and L are both null; Z2, wherein Z2 is agroup selected from:

wherein one of R1 and R2 is null and the other is as defined above, andp is an integer from 1 to 20; Z1-Z2, wherein Z1 and Z2 are as definedabove; the reactive moiety RM is independently null or a group selectedfrom:

wherein R4 and R5 are as defined above; R6 is C₁-C₃ alkyl or anelectron-withdrawing group, comprising NO₂ and CN group; r is an integerfrom 0 to 7; or a pharmaceutically acceptable salt thereof.
 3. Acompound according to claim 2, wherein Z is a group selected from:

wherein one of R1 and R2 is null and the other is as defined in claim 2;or a pharmaceutically acceptable salt thereof.
 4. A compound accordingto claim 1, or a pharmaceutically acceptable salt thereof, which isselected from the group consisting of:(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-ylglycinate;(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-ylL-phenylalanyl-L-leucylglycinate;(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-ylpiperazine-1-carboxylate;(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-ylN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-L-leucylglycinate;N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[4-({[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}carbonyl)piperazin-1-yl]carbonyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]piperazine-1-carboxylate;(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-ylmethyl[2-(methylamino)ethyl]carbamate;N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[{2-[({[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide;(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-amino-3,3-dimethylbutanoate hydrochloride;(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl5-amino-4,4-difluoropentanoate hydrochloride;4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl[1-(aminomethyl)cyclohexyl]acetate hydrochloride;L-valyl-N-[4-({[(5-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-difluoro-5-oxopentyl)carbamoyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide;L-valyl-N-[4-({[(4-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-dimethyl-4-oxobutyl)carbamoyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide;L-valyl-N-(4-{[({[1-(2-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2-oxoethyl)cyclohexyl]methyl}carbamoyl)oxy]methyl}phenyl)-N⁵-carbamoyl-L-ornithinamide;N-[6-(2,5-dioxopyrrolidin-1-yl)hexanoyl]-L-valyl-N-[4-({[(5-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-difluoro-5-oxopentyl)carbamoyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide;N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-(5-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-difluoro-5-oxopentyl)-N⁵-carbamoyl-L-ornithinamide;N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{[1-(2-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2-oxoethyl)cyclohexyl]methyl}-N⁵-carbamoyl-L-ornithinamide;N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-(4-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-dimethyl-4-oxobutyl)-N⁵-carbamoyl-L-ornithinamide;N-(3-carboxypropanoyl)-L-valyl-N-(5-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-difluoro-5-oxopentyl)-N⁵-carbamoyl-L-ornithinamide;N-(3-carboxypropanoyl)-L-valyl-N-{[1-(2-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2-oxoethyl)cyclohexyl]methyl}-N⁵-carbamoyl-L-ornithinamide;N-(3-carboxypropanoyl)-L-valyl-N-(4-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-dimethyl-4-oxobutyl)-N⁵-carbamoyl-L-ornithinamide;N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-N-(5-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-difluoro-5-oxopentyl)-L-leucinamide;N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-N-{[1-(2-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2-oxoethyl)cyclohexyl]methyl}-L-leucinamide;N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-N-(4-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-dimethyl-4-oxobutyl)-L-leucinamide;L-valyl-N-{[1-(2-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2-oxoethyl)cyclohexyl]methyl}-N⁵-carbamoyl-L-ornithinamide;L-valyl-N-(4-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-dimethyl-4-oxobutyl)-N⁵-carbamoyl-L-ornithinamide,andL-valyl-N-(5-{[(4S)-10-bromo-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-2,2-difluoro-5-oxopentyl)-N⁵-carbamoyl-L-ornithinamide.5. A pharmaceutical composition comprising a therapeutically effectiveamount of a compound of formula (I) or a pharmaceutically acceptablesalt thereof, as defined in claim 1, and at least one pharmaceuticallyacceptable excipient, carrier or diluent.
 6. A pharmaceuticalcomposition according to claim 5 further comprising one or morechemotherapeutic agents.
 7. A product comprising a compound of formula(I) or a pharmaceutically acceptable salt thereof, as defined in claim1, and one or more chemotherapeutic agents, as a combined preparationfor simultaneous, separate or sequential use in anticancer therapy.
 8. Acompound of formula (I) or a pharmaceutically acceptable salt thereof,as defined in claim 1, for use as a medicament.
 9. (canceled) 10.(canceled)
 11. A method for treating cancer, which comprisesadministering to a mammal in need thereof an effective amount of acompound of formula (I) or a pharmaceutically acceptable salt thereof,as defined in claim
 1. 12. A method according to claim 11, wherein themammal in need thereof is a human.
 13. (canceled)
 14. (canceled)
 15. Themethod according to claim 11, wherein the cancer is selected from thegroup consisting of carcinomas, such as bladder, breast, colon, kidney,liver, lung, including small cell lung cancer, esophagus, gall-bladder,ovary, pancreas, stomach, cervix, thyroid, prostate, and skin, includingsquamous cell carcinoma; hematopoietic tumors of lymphoid lineage,including leukemia, acute lymphocytic leukemia, acute lymphoblasticleukemia, B-cell lymphoma, T-cell-lymphoma, Hodgkin's lymphoma,non-Hodgkin's lymphoma, hairy cell lymphoma and Burkitt's lymphoma;hematopoietic tumors of myeloid lineage, including acute and chronicmyelogenous leukemias, myelodysplastic syndrome and promyelocyticleukemia; tumors of mesenchymal origin, including fibrosarcoma andrhabdomyosarcoma; tumors of the central and peripheral nervous system,including astrocytoma, neuroblastoma, glioma and schwannoma; othertumors, including melanoma, seminoma, teratocarcinoma, osteosarcoma,xeroderma pigmentosum, keratoxanthoma, thyroid follicular cancer,Kaposi's sarcoma and mesothelioma.